Caged neurotransmitters have emerged as a useful tool for the high-resolution, electrode-free chemical stimulation of single neurons or neural circuits. These probe compounds are prepared via covalent appendage of a light-sensitive protecting group—the cage—to a signaling molecule. FIG. 1 (top drawing) illustrates a single cage approach for uncaging the caged GABA compound. With the cage in place, the signaling molecule is intended to be unable to activate its receptor. Upon delivery of a pulse of light, the cage is rapidly cleaved to reveal the active signaling molecule. When introduced into sliced or intact living brain tissue, caged neurotransmitters may activate neurotransmitter pathways at defined locations with micron and millisecond precision. Because they act one level upstream from intracellular voltage and second messenger signaling, caged neurotransmitters were hoped to allow for a remarkable degree of specificity in chemical modulation of neural activity. It is possible to use patterned photostimulation techniques to achieve stimulation at many arbitrary locations in parallel; microelectrode-based methods are not amenable to this type of task (Katz, L. C.; Dalva, M. B. J. Neurosci. Meth. 1994, 54, 205-218; Shoham, S. et al., 3589601-1 Nat. Methods. 2005, 2, 837-843, which are incorporated herein by reference as if fully set forth). Moreover, neurotransmitter uncaging offers a useful alternative to optogenetic approaches because uncaging does not require gene delivery, is neurotransmitter-specific, and uses different wavelengths of light than those employed in optogenetics (Packer, A. M. et al., Nat. Neurosci. 2013, 16, 805-815, which is incorporated herein by reference as if fully set forth).
GABA (γ-amino butyric acid or gamma-amino butyric acid), is the chief vertebrate inhibitory neurotransmitter in mammals. It is an amino acid that contains an amino group and a carboxylic acid but due to the gamma-position of the amino group, GABA is not incorporated into proteins. GABA may be produced in inhibitory neurons from glutamate via glutamic acid decarboxylase (GAD), and may function as a neural inhibitor. GABA may agonize the GABA receptors, which act through these G-proteins to cause efflux of K+ and cause hyperpolarization of the cell (Purves et al., 2008 (Eds.). (2008). Neuroscience (4th ed.). Sunderland, Mass.: Sinauer Associates, Inc., which is incorporated herein by reference as if fully set forth). GABA may agonize GABA receptors that are not located only postsynaptically, but presynaptically as well. Presynaptic GABAB receptors are metabotropic receptors that suppress calcium influx, resulting in less neurotransmitter release and an overall inhibitory effect (Wang & Lambert, 1999 Journal of Neurophysiology 83, 1073-1078, which is incorporated herein by reference as if fully set forth).
A number of caged GABA—based compounds have been developed that satisfy many of these criteria: α-carboxy-2-nitrobenzyl (CNB)-, 4-carboxymethoxy-5,7-dinitroindolinyl (CDNI)-, 1,3-bis(dihydroxyphosphoryloxy)propan-2-yloxy]-7-nitroindoline (DPNI)-, 4-methoxy-5,7-dinitroindolinyl (MDNI)-, and ruthenium-bipyridine-triphenylphosphine-(RuBi-GABA) (Lester, H. A.; Nerbonne, J. M. Ann. Rev. Biophys. Bioeng. 1982, 11, 151-175; Adams, S. R.; Tsien, R. Y. Ann. Rev. Phys. 1993, 55, 755-784, which are incorporated herein as if fully set forth). These caged GABA compounds are chemically stable in aqueous solution on time scales of weeks or longer (Molnár, P.; Nadler, J. V. Eur. J. Pharmacol. 2000, 391, 255-262; Trigo, F. F. et al., J. Neurosci. Meth., 2009, 181, 159-169; Matsuzaki, M. et al., Nat. Chem. Biol. 2010, 6, 255-257, which are incorporated by reference as if fully set forth). However, they are not inactive at GABA receptors in their caged form. CNB-, CDNI-, DPNI-, and MDNI-caged GABA compounds are antagonists of GABAA receptors, as are RuBi-GABA, the related compound RuBi-glutamate, and 4-methoxy-7-nitroindolinyl (MNI)-glutamate (Molnár, P.; Nadler, J. V. Eur. J. Pharmacol. 2000, 391, 255-262; Trigo, F. F. et al., J. Neurosci. Meth., 2009, 181, 159-169; Matsuzaki, M. et al., Nat. Chem. Biol. 2010, 6, 255-257; Rial Verde, E. M. et al., Front. Neural Circuits. 2008, 2, 1-8; Fino, E. et al., Front. Neural Circuits, 2009, 3, 1-9, which are incorporated by reference as if fully set forth).
The practical limit at which these compounds can be used without interfering with neural circuit function is so low (<200 μM) that they cannot be used to attain the near-millimolar concentrations that occur locally during synaptic transmission (Perrais, D.; Ropert, N. J. Neurosci, 1999, 19, 578-588; Farrant, M.; Nusser, Z. Nat. Rev. Neurosci. 2005, 6, 215-229, both of which are incorporated by reference as if fully set forth).